An Experimental Model Of Hyperacute Rejection Of ABO-incompatible Renal Allograft In Nonhuman Primates

To establish a standard method using Flow Cytometry in ABO blood group antibody (Ab) detection. Based on direct hemagglutination, binding of antibody was measured by indirect flow cytometry adding secondary isotype-specific fluoresce labeled ab, For validation, we analyzed sera samples from 52 blood donors and compared with the results of using ELISA Reader method. Changes in antibody measured by both methods correlated well with sera titers. FCM showed better specificity, sensitivity and reproducibility over ELISA Reader method, and allowed quantitative discrimination in the range of antibody levels. FCM is the most objective method to offer accurate detection of Natural ABO blood group ab and more suitable for ABO-incompatible transplantations and pre-and post-transplant monitoring, that may replace the traditional hemagglutination.Like humans, most of non-human primates also express the ABH specificites of the ABO histo-blood group system. In this study, we therefore developed a simple and rapid method of measuring the levels and distribution of monkey ABO antibody (Ab) using flow cytometry (FCM). Binding of standard human A or B RBCs and anti-A/B Ab was measured by adding secondary isotype-specific fluoresce labeled Ab, for validation, we analyzed all collective monkey blood sera samples and compared with the results of human healthy blood donor sera. The results revealed that, following adsorption on human type O RBCs to remove anti-human antibodies, FCM seems to be the objective and reliable technique to detect natural specific Ab levels in Monkey sera. Both sera were detected and the Monkey’s anti-A/B antibody titers were significantly lower than Human’s (P<0.05). We concluded that FCM shows an excellent method to offer accurate detection of natural ABO blood group Ab on non-human primates, and also can be the technique support for the further study.Hyperacute rejection (HAR), which occurs in ABO-incompatible renal transplantation or xenotranplantation with high titers of natural anti-donor specific antigen antibody, is a significant cause of allografts dysfunction and loss. More efficacious treatment modalities are needed to eliminate or curtail natural anti-ABO blood group antibody production and its deleterious effects on the kidney. The availability of animal models mimicking human HAR is essential to understand its pathophysiology and develop new treatment strategies. Using a non-human primate’s ABO-incompatible renal transplant model, we demonstrate that presensitization of recipients with KLH-A results in rejection of subsequent renal allografts. All presensitized monkeys (n=2) developed renal failure within the graft minutes to a few hours after activation of complement, with the vascular anastomosis, with subsequent arteriographic evidence of progressive cortical necrosis, necessitating removal of the grafts. The major histological findings that are associated with HAR are neutrophil and platelet margination in glomerular and peritubular capillaries, red blood cells stasis, fibrin deposition, and thrombosis within the microvasculature with sparing of larger blood vessels (showed in 07cy05; at least early in the process), and acute tubular injury and variable levels of cortical necrosis. Immunofluorescence (IF) studies demonstrated IgM (but not IgG) combination in glomerular and peritubular capillaries. Complement (C4d or C5b-9) deposition was diffuse and obvious in peritubular capillaries. Serum analysis demonstrated high levels of circulating anti-ABO blood group antibodies with strongly reactivity to blood group antigen. The clinical setting and histological findings of our models strongly resemble HAR, which was frequently associated with humoral rejection, a situation commonly encountered in human renal allograft recipients. These findings in our model established for HAR indicate that this non-human primate’s model may provide an excellent tool to explore the pathogenesis and immunological events involved in human renal allograft rejection. The development of this valuable model has the potential to advance understanding in several key areas including local and systemic immunological events that result in natural blood group antibody elaboration, its contribution to graft injury, and the effects of various potential therapeutic interventions, mechanisms of natural blood group antibody-mediated graft damage, and effective treatment strategies to interfere with these processes.